Expression of an MSP1 multi-epitope protein from Anaplasma marginale for the analysis of immune responses against multimeric proteins

The outer membrane proteins (OMPs) of bacterial pathogens interact with the host, including the immune system. This interaction is often dependant on conformation and the association between individual OMPs. In Anaplasma marginale covalent and non-covalent interactions amongst the six major surface proteins, MSP1a, MSP1b and MSP2-5 have been shown. Because of the extensive interaction between the OMPs it is not surprising that a single protein does not fully protect against disease. The MSP1 complex, formed by covalently linked MSP1a and MSP1b, significantly protects against disease following challenge, although unassociated recombinant MSP1a and MSP1b do not. Our goal is to understand the role complexed proteins play in eliciting protective immune responses. In cattle immunized with the MSP1 complex, antibodies against MSP1a and MSP1b are elicited, although CD4+ T-cell epitopes were predominantly identified for MSP1a. We hypothesized that MSP1a-specific CD4+ T-cells provide help to B-cells specific for MSP1b as well as MSP1a. A construct with the MSP1a T-cell epitopes linked to MSP1b was generated by a strategy combined of assembly PCR and restriction enzyme cloning. This and unlinked constructs will be used to immunize cattle to determine the requirement for linked recognition of T and B cell epitopes on MSP1 for generating protective immune responses.